Mouse microglia express unique miRNA-mRNA networks to facilitate age-specific functions in the developing central nervous system.

Microglia regulate multiple processes in the central nervous system, exhibiting a considerable level of cellular plasticity which is facilitated by an equally dynamic transcriptional environment. While many gene networks that regulate microglial functions have been characterised, the influence of epigenetic regulators such as small non-coding microRNAs (miRNAs) is less well defined. We have sequenced the miRNAome and mRNAome of mouse microglia during brain development and adult homeostasis, identifying unique profiles of known and novel miRNAs. Microglia express both a consistently enriched miRNA signature as well as temporally distinctive subsets of miRNAs. We generated robust miRNA-mRNA networks related to fundamental developmental processes, in addition to networks associated with immune function and dysregulated disease states. There was no apparent influence of sex on miRNA expression. This study reveals a unique developmental trajectory of miRNA expression in microglia during critical stages of CNS development, establishing miRNAs as important modulators of microglial phenotype.

Detection of PERK Signaling in the Central Nervous System.

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) signaling adapts cells to stressful conditions by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). Phosphorylation of eIF2α inhibits global protein translation but stimulates the expression of numerous stress-responsive genes by inducing the transcription factor ATF4. A large number of studies have shown that activation of PERK signaling has beneficial or detrimental effects in various diseases of the central nervous system (CNS), including neurodegenerative diseases, myelin disorders, CNS injuries, among others. This chapter is devoted to describing the practical methods for the detection of PERK signaling in CNS diseases.

Endoplasmic reticulum associated degradation is required for maintaining endoplasmic reticulum homeostasis and viability of mature Schwann cells in adults.

The integrated unfolded protein response (UPR) and endoplasmic reticulum associated degradation (ERAD) is the principle mechanisms that maintain endoplasmic reticulum (ER) homeostasis. Schwann cells (SCs) must produce an enormous amount of myelin proteins via the ER to assemble and maintain myelin structure; however, it is unclear how SCs maintain ER homeostasis. It is known that Suppressor/Enhancer of Lin-12-like (Sel1L) is necessary for the ERAD activity of the Sel1L- hydroxymethylglutaryl reductase degradation protein 1(Hrd1) complex. Herein, we showed that Sel1L deficiency in SCs impaired the ERAD activity of the Sel1L-Hrd1 complex and led to ER stress and activation of the UPR. Interestingly, Sel1L deficiency had no effect on actively myelinating SCs during development, but led to later-onset mature SC apoptosis and demyelination in the adult PNS. Moreover, inactivation of the pancreatic ER kinase (PERK) branch of the UPR did not influence the viability and function of actively myelinating SCs, but resulted in exacerbation of ER stress and apoptosis of mature SCs in SC-specific Sel1L deficient mice. These findings suggest that the integrated UPR and ERAD is dispensable to actively myelinating SCs during development, but is necessary for maintaining ER homeostasis and the viability and function of mature SCs in adults.

The Integrated UPR and ERAD in Oligodendrocytes Maintain Myelin Thickness in Adults by Regulating Myelin Protein Translation.

Myelin proteins, which are produced in the endoplasmic reticulum (ER), are essential and necessary for maintaining myelin structure. The integrated unfold protein response (UPR) and ER-associated degradation (ERAD) are the primary ER quality control mechanism. The adaptor protein Sel1L (Suppressor/Enhancer of Lin-12-like) controls the stability of the E3 ubiquitin ligase Hrd1 (hydroxymethylglutaryl reductase degradation protein 1), and is necessary for the ERAD activity of the Sel1L-Hrd1 complex. Herein, we showed that Sel1L deficiency specifically in oligodendrocytes caused ERAD impairment, the UPR activation, and attenuation of myelin protein biosynthesis; and resulted in late-onset, progressive myelin thinning in the CNS of adult mice (both male and female). The pancreatic ER kinase (PERK) branch of the UPR functions as the master regulator of protein translation in ER-stressed cells. Importantly, PERK inactivation reversed attenuation of myelin protein biosynthesis in oligodendrocytes and restored myelin thickness in the CNS of oligodendrocyte-specific Sel1L-deficient mice (both male and female). Conversely, blockage of proteolipid protein production exacerbated myelin thinning in the CNS of oligodendrocyte-specific Sel1L-deficient mice (both male and female). These findings suggest that impaired ERAD in oligodendrocytes reduces myelin thickness in the adult CNS through suppression of myelin protein translation by activating PERK.SIGNIFICANCE STATEMENT Myelin is an enormous extended plasma membrane of oligodendrocytes that wraps and insulates axons. Myelin structure, including thickness, was thought to be extraordinarily stable in adults. Myelin proteins, which are produced in the endoplasmic reticulum (ER), are essential and necessary for maintaining myelin structure. The integrated unfolded protein response (UPR) and ER-associated degradation (ERAD) are the primary mechanism that maintains ER protein homeostasis. Herein, we explored the role of the integrated UPR and ERAD in oligodendrocytes in regulating myelin protein production and maintaining myelin structure using mouse models. The results presented in this study imply that the integrated UPR and ERAD in oligodendrocytes maintain myelin thickness in adults by regulating myelin protein production.

NF-κB Activation Accounts for the Cytoprotective Effects of PERK Activation on Oligodendrocytes during EAE.

Previous studies demonstrate that activation of pancreatic ER kinase (PERK) protects oligodendrocytes against inflammation in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Interestingly, data indicate that the cytoprotective effects of PERK activation on oligodendrocytes during EAE are not mediated by activating transcription factor 4 (ATF4) but are accompanied by activation of nuclear factor κB (NF-κB). NF-κB plays a critical role in MS and EAE; however, the effects of NF-κB activation on oligodendrocytes in these diseases remain elusive. Herein, we generated a mouse model that allow for activation of NF-κB specifically in oligodendrocytes and found that enhanced NF-κB activation in oligodendrocytes had a minimal effect on their viability and function under normal conditions (both male and female mice). Interestingly, we found that enhanced NF-κB activation in oligodendrocytes attenuated EAE disease severity and ameliorated EAE-induced oligodendrocyte loss, demyelination, and axon degeneration, without affecting inflammation (female mice). Moreover, we showed that the detrimental effects of PERK inactivation in oligodendrocytes in EAE were accompanied by impaired NF-κB activation in oligodendrocytes, and were completely rescued by enhanced NF-κB activation in oligodendrocytes (female mice). These findings suggest that NF-κB activation accounts for the cytoprotective effects of PERK activation on oligodendrocytes in MS and EAE.SIGNIFICANCE STATEMENT Nuclear factor κB (NF-κB) is activated in oligodendrocytes in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE); however, the role of NF-κB activation in oligodendrocytes in MS and EAE remains elusive. Herein, we generated a mouse model that allows for activation of NF-κB selectively in oligodendrocytes and demonstrated that NF-κB activation prevented oligodendrocyte death and myelin damage in the EAE model. We further demonstrated that NF-κB activation contributed to the protective effects of pancreatic ER kinase (PERK) activation on oligodendrocytes in the EAE model. As such, this work will facilitate the development of new treatments that enhance oligodendrocyte survival in MS patients by targeting the PERK-NF-κB pathway.

The UPR preserves mature oligodendrocyte viability and function in adults by regulating autophagy of PLP.

Maintaining cellular proteostasis is essential for oligodendrocyte viability and function; however, its underlying mechanisms remain unexplored. Unfolded protein response (UPR), which comprises 3 parallel branches, inositol requiring enzyme 1 (IRE1), pancreatic ER kinase (PERK), and activating transcription factor 6α (ATF6α), is a major mechanism that maintains cellular proteostasis by facilitating protein folding, attenuating protein translation, and enhancing autophagy and ER-associated degradation. Here we report that impaired UPR in oligodendrocytes via deletion of PERK and ATF6α did not affect developmental myelination but caused late-onset mature oligodendrocyte dysfunction and death in young adult mice. The detrimental effects of the impaired UPR on mature oligodendrocytes were accompanied by autophagy impairment and intracellular proteolipid protein (PLP) accumulation and were rescued by PLP deletion. Data indicate that PLP was degraded by autophagy and that intracellular PLP accumulation was cytotoxic to oligodendrocytes. Thus, these findings imply that the UPR is required for maintaining cellular proteostasis and the viability and function of mature oligodendrocytes in adults by regulating autophagy of PLP.

Unfolded protein response in myelin disorders.

Activation of the unfolded protein response in response to endoplasmic reticulum stress preserves cell viability and function under stressful conditions. Nevertheless, persistent, unresolvable activation of the unfolded protein response can trigger apoptosis to eliminate stressed cells. Recent studies show that the unfolded protein response plays an important role in the pathogenesis of various disorders of myelin, including multiples sclerosis, Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease, vanishing white matter disease, spinal cord injury, tuberous sclerosis complex, and hypoxia-induced perinatal white matter injury. In this review we summarize the current literature on the unfolded protein response and the evidence for its role in the pathogenesis of myelin disorders.

Neuron-specific PERK inactivation exacerbates neurodegeneration during experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are chronic inflammatory demyelinating and neurodegenerative diseases of the CNS. Although neurodegeneration is the major contributor to chronic disability in MS, mechanisms governing the viability of axons and neurons in MS and EAE remain elusive. Data indicate that activation of pancreatic endoplasmic reticulum kinase (PERK) influences, positively or negatively, neuron and axon viability in various neurodegenerative diseases through induction of ATF4. In this study, we demonstrate that the PERK pathway was activated in neurons during EAE. We found that neuron-specific PERK inactivation impaired EAE resolution and exacerbated EAE-induced axon degeneration, neuron loss, and demyelination. Surprisingly, neuron-specific ATF4 inactivation did not alter EAE disease course or EAE-induced axon degeneration, neuron loss, and demyelination. These results suggest that PERK activation in neurons protects axons and neurons against inflammation in MS and EAE through ATF4-independent mechanisms.

Role of nuclear factor κB in multiple sclerosis and experimental autoimmune encephalomyelitis.

The transcription factor nuclear factor κB (NF-κB) plays major roles in inflammatory diseases through regulation of inflammation and cell viability. Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). It has been shown that NF-κB is activated in multiple cell types in the CNS of MS patients, including T cells, microglia/macrophages, astrocytes, oligodendrocytes, and neurons. Interestingly, data from animal model studies, particularly studies of experimental autoimmune encephalomyelitis, have suggested that NF-κB activation in these individual cell types has distinct effects on the development of MS. In this review, we will cover the current literature on NF-κB and the evidence for its role in the development of MS and its animal model experimental autoimmune encephalomyelitis.

Activating transcription factor 6α deficiency exacerbates oligodendrocyte death and myelin damage in immune-mediated demyelinating diseases.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) play a critical role in immune-mediated demyelinating diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), by regulating the viability of oligodendrocytes. Our previous studies show that activation of the PERK branch of the UPR protects myelinating oligodendrocytes against ER stress in young, developing mice that express IFN-γ, a key pro-inflammatory cytokine in MS and EAE, in the CNS. Several studies also demonstrate that PERK activation preserves oligodendrocyte viability and function, protecting mice against EAE. While evidence suggests activation of the ATF6α branch of the UPR in oligodendrocytes under normal and disease conditions, the effects of ATF6α activation on oligodendrocytes in immune-mediated demyelinating diseases remain unknown. Herein, we showed that ATF6α deficiency had no effect on oligodendrocytes under normal conditions. Interestingly, we showed that ATF6α deficiency exacerbated ER stressed-induced myelinating oligodendrocyte death and subsequent myelin loss in the developing CNS of IFN-γ-expressing mice. Moreover, we found that ATF6α deficiency increased EAE severity and aggravated EAE-induced oligodendrocyte loss and demyelination, without affecting inflammation. Thus, these data suggest the protective effects of ATF6α activation on oligodendrocytes in immune-mediated demyelinating diseases.

NF-κB Activation Protects Oligodendrocytes against Inflammation.

NF-κB is a key player in inflammatory diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the effects of NF-κB activation on oligodendrocytes in MS and EAE remain unknown. We generated a mouse model that expresses IκBαΔN, a super-suppressor of NF-κB, specifically in oligodendrocytes and demonstrated that IκBαΔN expression had no effect on oligodendrocytes under normal conditions (both sexes). Interestingly, we showed that oligodendrocyte-specific expression of IκBαΔN blocked NF-κB activation in oligodendrocytes and resulted in exacerbated oligodendrocyte death and hypomyelination in young, developing mice that express IFN-γ ectopically in the CNS (both sexes). We also showed that NF-κB inactivation in oligodendrocytes aggravated IFN-γ-induced remyelinating oligodendrocyte death and remyelination failure in the cuprizone model (male mice). Moreover, we found that NF-κB inactivation in oligodendrocytes increased the susceptibility of mice to EAE (female mice). These findings imply the cytoprotective effects of NF-κB activation on oligodendrocytes in MS and EAE.SIGNIFICANCE STATEMENT Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. NF-κB is a major player in inflammatory diseases that acts by regulating inflammation and cell viability. Data indicate that NF-κB activation in inflammatory cells facilitates the development of MS. However, to date, attempts to understand the role of NF-κB activation in oligodendrocytes in MS have been unsuccessful. Herein, we generated a mouse model that allows for inactivation of NF-κB specifically in oligodendrocytes and then used this model to determine the precise role of NF-κB activation in oligodendrocytes in models of MS. The results presented in this study represent the first demonstration that NF-κB activation acts cell autonomously to protect oligodendrocytes against inflammation in animal models of MS.

Dual role of the integrated stress response in medulloblastoma tumorigenesis.

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). Phosphorylated eIF2α is quickly dephosphorylated by the protein phosphatase 1 and growth arrest and DNA damage 34 (GADD34) complex. Data indicate that the ISR can either promote or suppress tumor development. Our previous studies showed that the ISR is activated in medulloblastoma in both human patients and animal models, and that the decreased ISR via PERK heterozygous deficiency attenuates medulloblastoma formation in Patched1 heterozygous deficient (Ptch1+/-) mice by enhancing apoptosis of pre-malignant granule cell precursors (GCPs) during cell transformation. We showed here that GADD34 heterozygous mutation moderately enhanced the ISR and noticeably increased the incidence of medulloblastoma in adult Ptch1+/- mice. Surprisingly, GADD34 homozygous mutation strongly enhanced the ISR, but significantly decreased the incidence of medulloblastoma in adult Ptch1+/- mice. Intriguingly, GADD34 homozygous mutation significantly enhanced pre-malignant GCP apoptosis in cerebellar hyperplastic lesions and reduced the lesion numbers in young Ptch1+/- mice. Nevertheless, neither GADD34 heterozygous mutation nor GADD34 homozygous mutation had a significant effect on medulloblastoma cells in adult Ptch1+/- mice. Collectively, these data imply the dual role of the ISR, promoting and inhibiting, in medulloblastoma tumorigenesis by regulating apoptosis of pre-malignant GCPs during the course of malignant transformation.

Type II Activation of Macrophages and Microglia by Immune Complexes Enhances Th17 Biasing in an IL-6-Independent Manner.

Macrophages can be activated into several distinct activation states. One of these states, type II activation, has a regulatory phenotype characterized by decreased IL-12 and increased IL-10, and has been shown to bias naïve CD4+ T cells to a Th2 response. Microglia, the resident macrophage-like cells in the central nervous system (CNS), are important contributors to neuroinflammation and, thus, we investigated if type II activated microglia could bias CD4+ T cell responses in a similar manner as type II activated macrophages. Using immune complex ligation in the presence of LPS to induce type II activation, we found that both type II macrophages and type II microglia biased CD4+ T cell responses in vitro to express increased levels of IL-17A and CD124. The enhanced IL-17A production occurred independently of IL-6, and IL-10 and IL-12, which were key regulators of IFN-γ production, but were not involved in the increased IL-17A. Finally, we found that another type II-activating compound, glatiramer acetate, did not bias CD4+ T cells to produce enhanced IL-17A. Taken together, this study demonstrates that microglia can be type II activated and, similarly to type II macrophages, can bias CD4+ T cell responses; however, depending on the type II stimulus, the effect on CD4+ T cell subset differentiation may vary.

Inhibition of Vascular Endothelial Growth Factor Receptor 2 Exacerbates Loss of Lower Motor Neurons and Axons during Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are inflammatory demyelinating and neurodegenerative diseases in the central nervous system (CNS). It is believed that MS and EAE are initiated by autoreactive T lymphocytes that recognize myelin antigens; however, the mechanisms responsible for neurodegeneration in these diseases remain elusive. Data indicate that vascular endothelial growth factor A (VEGF-A) plays a role in the development of MS and EAE. Interestingly, VEGF-A is regarded as a neurotrophic factor in the CNS that promotes neuron survival and neurogenesis in various neurodegenerative diseases by activating VEGF receptor 2 (VEGFR2). In this study, we sought to explore the role of the VEGF-A/VEGFR2 signaling in neurodegeneration in MS and EAE. We showed that the expression of VEGF-A was decreased in the spinal cord during EAE and that VEGFR2 was activated in lower motor neurons in the spinal cord of EAE mice. Interestingly, we found that treatment with SU5416, a selective VEGFR2 inhibitor, starting after the onset of EAE clinical symptoms exacerbated lower motor neuron loss and axon loss in the lumbar spinal cord of mice undergoing EAE, but did not alter Purkinje neuron loss in the cerebellum or upper motor neuron loss in the cerebral cortex. Moreover, SU5416 treatment had a minimal effect on EAE clinical symptoms as well as inflammation, demyelination, and oligodendrocyte loss in the lumbar spinal cord. These results imply the protective effects of the VEGF-A/VEGFR2 signaling on lower motor neurons and axons in the spinal cord in MS and EAE.

The unfolded protein response in multiple sclerosis.

The unfolded protein response (UPR) occurs in response to endoplasmic reticulum (ER) stress caused by the accumulation of unfolded or misfolded proteins in the ER. The UPR is comprised of three signaling pathways that promote cytoprotective functions to correct ER stress; however, if ER stress cannot be resolved the UPR results in apoptosis of affected cells. The UPR is an important feature of various human diseases, including multiple sclerosis (MS). Recent studies have shown several components of the UPR are upregulated in the multiple cell types in MS lesions, including oligodendrocytes, T cells, microglia/macrophages, and astrocytes. Data from animal model studies, particularly studies of experimental autoimmune encephalomyelitis (EAE) and the cuprizone model, imply an important role of the UPR activation in oligodendrocytes in the development of MS. In this review we will cover current literature on the UPR and the evidence for its role in the development of MS.

Enhanced immunosuppression by therapy-exposed glioblastoma multiforme tumor cells.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an extremely short time to relapse following standard treatment. Since recurrent GBM is often resistant to subsequent radiotherapy and chemotherapy, immunotherapy has been proposed as an alternative treatment option. Although it is well established that GBM induces immune suppression, it is currently unclear what impact prior conventional therapy has on the ability of GBM cells to modulate the immune environment. In this study, we investigated the interaction between immune cells and glioma cells that had been exposed to chemotherapy or irradiation in vitro. We demonstrate that treated glioma cells are more immunosuppressive than untreated cells and form tumors at a faster rate in vivo in an animal model. Cultured supernatant from in vitro-treated primary human GBM cells were also shown to increase suppression, which was independent of accessory suppressor cells or T regulatory cell generation, and could act directly on CD4(+) and CD8(+) T cell proliferation. While a number of key immunosuppressive cytokines were overexpressed in the treated cells, including IL-10, IL-6 and GM-CSF, suppression could be alleviated in a number of treated GBM lines by inhibition of prostaglandin E2. These results reveal for the first time that conventional therapies can alter immunosuppressive pathways in GBM tumor cells, a finding with important implications for the combination of immunotherapy with standard treatment.

Treatment with the antipsychotic agent, risperidone, reduces disease severity in experimental autoimmune encephalomyelitis.

Recent studies have demonstrated that atypical antipsychotic agents, which are known to antagonize dopamine D2 and serotonin 5-HT2a receptors, have immunomodulatory properties. Given the potential of these drugs to modulate the immune system both peripherally and within the central nervous system, we investigated the ability of the atypical anti-psychotic agent, risperidone, to modify disease in the animal model of multiple sclerosis (MS)4, experimental autoimune encephalomyelitis (EAE). We found that chronic oral administration of risperidone dose-dependently reduced the severity of disease and decreased both the size and number of spinal cord lesions. Furthermore, risperidone treatment substantially reduced antigen-specific interleukin (IL)-17a, IL-2, and IL-4 but not interferon (IFN)-γ production by splenocytes at peak disease and using an in vitro model, we show that treatment of macrophages with risperidone alters their ability to bias naïve T cells. Another atypical antipsychotic agent, clozapine, showed a similar ability to modify macrophages in vitro and to reduce disease in the EAE model but this effect was not due to antagonism of the type 1 or type 2 dopamine receptors alone. Finally, we found that while risperidone treatment had little effect on the in vivo activation of splenic macrophages during EAE, it significantly reduced the activation of microglia and macrophages in the central nervous system. Together these studies indicate that atypical antipsychotic agents like risperidone are effective immunomodulatory agents with the potential to treat immune-mediated diseases such as MS.

Targeting innate receptors with MIS416 reshapes Th responses and suppresses CNS disease in a mouse model of multiple sclerosis.

Modification of the innate immune cell environment has recently been recognized as a viable treatment strategy for reducing autoimmune disease pathology. MIS416 is a microparticulate immune response modifier that targets myeloid cells, activating cytosolic receptors NOD2 and TLR9, and has completed a phase 1b/2a trial for the treatment of secondary progressive multiple sclerosis. Using a mouse model of multiple sclerosis, we are investigating the pathways by which activation of TLR9 and NOD2 may modify the innate immune environment and the subsequent T cell-mediated autoimmune responses. We have found that MIS416 has profound effects on the Th subset balance by depressing antigen-specific Th1, Th17, and Th2 development. These effects coincided with an expansion of specific myeloid subpopulations and increased levels of MIS416-stimulated IFN-γ by splenocytes. Additionally, systemic IFN-γ serum levels were enhanced and correlated strongly with disease reduction, and the protective effect of MIS416 was abrogated in IFN-γ-deficient animals. Finally, treatment of secondary progressive MS patients with MIS416 similarly elevated the levels of IFN-γ and IFN-γ-associated proteins in the serum. Together, these studies demonstrate that administration of MIS416, which targets innate cells, reshapes autoimmune T cell responses and leads to a significant reduction in CNS inflammation and disease.

Type II-activated murine macrophages produce IL-4.

BACKGROUND: Type II activation of macrophages is known to support Th2 responses development; however, the role of Th2 cytokines (esp. IL-4) on type II activation is unknown. To assess whether the central Th2 cytokine IL-4 can alter type II activation of macrophages, we compared the ability of bone marrow-derived macrophages from wild type (WT) and IL-4Rα-deficient mice to be classically or type II-activated in vitro. RESULTS: We found that although both WT and IL-4Rα-deficient macrophages could be classically activated by LPS or type II activated by immune complexes plus LPS, IL-4Rα-deficient macrophages consistently produced much higher levels of IL-12p40 and IL-10 than WT macrophages. Additionally, we discovered that type II macrophages from both strains were capable of producing IL-4; however, this IL-4 was not responsible for the reduced IL-12p40 and IL-10 levels produced by WT mice. Instead, we found that derivation culture conditions (GM-CSF plus IL-3 versus M-CSF) could explain the different responses of BALB/c and IL-4Rα-/- macrophages, and these cytokines shaped the ensuing macrophage such that GM-CSF plus IL-3 promoted more IL-12 and IL-4 while M-CSF led to higher IL-10 production. Finally, we found that enhanced IL-4 production is characteristic of the type II activation state as other type II-activating products showed similar results. CONCLUSIONS: Taken together, these results implicate type II activated macrophages as an important innate immune source of IL-4 that may play an important role in shaping adaptive immune responses.